AChR activation (function and structure)

A. cyclic activation scheme. Vertical, gating. Receptors isomerize between resting (C) and active (O) conformations (equilibrium constant Ln, where n is the number of bound ligands). Horizontal, binding. Agonists (A) bind weakly to C (equilibrium dissociation constant KdC, free energy change ΔGLA) and strongly to O (KdO, ΔGLA). In adult AChRs the 2 sites are equivalent and independent and there is no significant external energy L2/L0 =(KdC/KdO)2 (Nayak & Auerbach, 2017). B. intermediate steps in binding and gating. Left, the agonist diffuses to the target to form an encounter complex that then becomes a LA complex by a ‘catch’ rearrangement. Right, The LA site becomes HA by a ‘hold’ local rearrangement, followed by domain restructurings that eventually generate a conducting pore. Bold, the. free energy changes in catch and hold (ΔGLA and ΔGHA) determine agonist action. C. overlay of α and δ subunits extracellular domains, toxin-less (red, 6UWX.pdb) and apo (blue, 7QKO.pdb). There are no major deviations (Cα RMSD = 0.3 Å). D. α-δ subunit neurotransmitter site with CCh (blue) in a HA, desensitized conformation (7QL6.pdb; (Zarkadas et al., 2022)). The agonist is in the trans orientation (tail points towards δ).

Agonist docking and dynamics

A. top, Agonist structures (blue, main N+): carbamylcholine (CCh), acetylcholine (ACh), epibatidine (Ebt) and epiboxidine (Ebx). B. top, root-mean-square-deviation (RMSD) of the system Cα (mean±SD, triplicates) throughout 200 ns MD simulations (ACh, cyan; CCh, green; Ebt, orange; Ebx, purple). bottom, close-up view of the pocket showing movements during MD simulations with CCh (red, apo; orange, 0 ns; blue, 200 ns;). Loop C moves down, loop F closes in, the agonist rotates cis→trans. C. α-Δ binding pocket (6UVW.pdb minus toxin) with agonists (top 3 poses). top, docked into the 0 ns structure (red). Loop C is up, loop F is out, agonist is cis. bottom, docked into the 200 ns structure after removing CCh (blue). Loop C is down, loop F is in and the agonist is trans. D. Docking score energies (mean±SD), 0 ns (red) and 200 ns (blue) suggest a LA→HA transition during the course of the simulation.

Principal Component Analysis (PCA)

For each agonist, the left panel plots PC1 vs PC2, the first two principal components that capture the maximum variance in the trajectory. Colors represent free energy value in kcal/mol (upper left, bottom). For all agonists there are 3 energy minima (red), m1, m2, and m3, that correspond to different conformations of the protein-ligand complex. The right panels are ‘porcupine’ plots that indicate the direction and magnitude of changes between PC1 and PC2. In order of appearance, m1 (≤20 ns)< m2<m3 (≥180 ns) (see text). m1 represents ACLA, m3 represents ACHA, and m2 is an intermediate configuration (corresponding ΔGs given in Table 1).

Calculated and experimental binding energies (kcal/mol)

Binding free energies and pocket properties

A. Calculated Gibbs free energy (ΔG) using MM-PBSA for the three distinct energy minima (m1, m2, and m3) showing the progression LA to HA (agonists, Fig. 2A). ΔGm2 is intermediate for ACh and CCh but least stable for Ebt and Ebx. B. vdW interactions increase m1 (ACLA) to m3 (ACHA), consistent with increased hydrophobicity. C. Binding pocket volume reduces m1 to m3, reflecting the more tightly packed pocket in ACHA. D. The number of water molecules in the pocket decreases m1→m3, orange, CLA; blue, CHA, reflecting a more hydrophobic environment in ACHA.

Agonist and loop movements in hold

In each panel, left, superimposed cartoons of ACLA (m1; orange) and ACHA (m3; blue). Loop C is upper left and loop F is lower right. In ACLA→ACHA (orange→blue) there is a cis→trans reorientation of the agonist (a flip) and a downward movement of loop C (a flop). right, the degree of flip from m1 (red) to m2 (yellow) to m3 (blue). The range, m1 (ACLA) →m3 (ACHA), is 131° to 176°.

Representative snapshots in hold

left, rearrangements of loop C, loop F and the ligand (m1, red; m2, yellow; m3, blue). Right, conserved residues and ligand orientation. m1 is ACLA and m3 is ACHA. In m1, a functional group at the agonist tail interact with αY93 (all agonists) and αD200 (CCh and Ebt). The position and orientation of αW149 relative to N+ of the agonist remains unchanged m1→m3 and serves as a fulcrum for the cis→trans flip (see Fig. 5). In m2, the functional nitrogen at the agonist tail (CCh, Ebt and Ebx) interacts with the hydroxyl group of αY198. For all ligands, loop C flop (m1→m3) repositions αY190 in pocket. In m3, the agonist fully flips to trans, facilitating the interaction of N+ with αY190 and the formation of water-mediated hydrogen bond with the reactive group at the tail with δN109/δL121 (loop E).